U.S. patent application number 13/575734 was filed with the patent office on 2012-11-29 for spectrophotofluorometer and fluorescence detector for liquid chromatograph.
Invention is credited to Izumi Ogata.
Application Number | 20120298881 13/575734 |
Document ID | / |
Family ID | 44318782 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120298881 |
Kind Code |
A1 |
Ogata; Izumi |
November 29, 2012 |
SPECTROPHOTOFLUOROMETER AND FLUORESCENCE DETECTOR FOR LIQUID
CHROMATOGRAPH
Abstract
Disclosed is a spectrophotofluorometer, which can shorten a
measuring time by efficiently obtaining a three-dimensional
spectral disposition, reduce sample deterioration and reduce the
size of the obtained data. The spectrophotofluorometer is provided
with a sample cell housing a sample, the components of which are
analyzed; an excitation light side spectroscope for irradiating
onto the sample cell excitation light with a predetermined
wavelength; a fluorescence side spectroscope for dispersing the
fluorescence from the sample cell by scanning a predetermined range
of wavelength; a fluorescence detector for detecting the
fluorescence from the fluorescence side spectroscope; and a
computer for obtaining a three-dimensional spectral disposition of
the fluorescence intensity in the sample on the basis of the
wavelength and the intensity of the fluorescence detected by the
fluorescence detector while changing the wavelength of the
excitation light irradiated onto the sample cell by the excitation
light side spectroscope. The computer sets a plurality of
combinations of the range of wavelengths of the excitation light
dispersed by the excitation light side spectroscope and the range
of wavelengths of the fluorescence dispersed by the fluorescence
side spectroscope.
Inventors: |
Ogata; Izumi; (Mito,
JP) |
Family ID: |
44318782 |
Appl. No.: |
13/575734 |
Filed: |
November 4, 2010 |
PCT Filed: |
November 4, 2010 |
PCT NO: |
PCT/JP2010/006478 |
371 Date: |
July 27, 2012 |
Current U.S.
Class: |
250/432R |
Current CPC
Class: |
G01N 30/74 20130101;
G01J 3/4406 20130101; G01J 3/12 20130101; G01N 2021/6423 20130101;
G01N 30/74 20130101; G01N 2021/6423 20130101; G01N 21/645 20130101;
G01N 2021/6417 20130101 |
Class at
Publication: |
250/432.R |
International
Class: |
G01J 3/443 20060101
G01J003/443 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2010 |
JP |
2010-016152 |
Claims
1. A fluorescence spectrophotometer comprising: a sample cell
housing a sample, components of the sample being analyzed; an
excitation light side spectroscope irradiating excitation light of
a predetermined wavelength onto the sample cell; an emission side
spectroscope dispersing emission light from the sample cell by
scanning a predetermined range of wavelengths; an emission light
detector detecting the emission light from the emission side
spectroscope; and a computer calculating a three-dimensional
spectrum of an emission light intensity in the sample on the basis
of a wavelength and an intensity of the emission light detected by
the emission light detector while changing a wavelength of the
excitation light irradiated onto the sample cell by the excitation
light side spectroscope, wherein the computer employs a plurality
of combinations of a range of wavelengths of the excitation light
dispersed by the excitation light side spectroscope and a range of
wavelengths of the emission light dispersed by the emission side
spectroscope.
2. The fluorescence spectrophotometer according to claim 1, wherein
the computer calculates the three-dimensional spectrum of the
emission light intensity in the sample on the basis of the set
combinations of the range of wavelengths of the excitation light
dispersed by the excitation light side spectroscope and the range
of wavelengths of the emission light dispersed by the emission side
spectroscope.
3. The fluorescence spectrophotometer according to claim 1, further
comprising a storage section storing a data group composed of data
of the three-dimensional spectrum calculated with respect to each
of a plurality of combination sets, each of the combination sets
formed by combining the range of wavelengths of the excitation
light dispersed by the excitation light side spectroscope and the
range of wavelengths of the emission light dispersed by the
emission side spectroscope.
4. The fluorescence spectrophotometer according to claim 3, wherein
the data of the three-dimensional spectrum is stored in a cell of
the storage section.
5. The fluorescence spectrophotometer according to claim 4, wherein
a value indicating that no data is provided is stored in a cell of
the storage section excluding a cell, the calculated data of the
three-dimensional spectrum being stored in the cell.
6. The fluorescence spectrophotometer according to claim 4, wherein
a value indicating that no measurement has been performed is stored
in a cell corresponding to an unmeasured wavelength region of the
storage section.
7. A fluorescence detector for a liquid chromatograph for use in a
liquid chromatograph injecting a sample into an eluent, separating
the sample into components in a separation column, and analyzing
the components of the sample by detecting the components, the
emission light detector comprising: a sample cell, the components
of the sample flowing through the sample cell; an excitation light
side spectroscope irradiating excitation light of a predetermined
wavelength onto the sample cell; an emission side spectroscope
dispersing emission light from the sample cell by scanning a
predetermined range of wavelengths; an emission light detector
detecting the emission light from the emission side spectroscope;
and a computer calculating a three-dimensional spectrum of an
emission light intensity in the sample on the basis of a wavelength
and an intensity of the emission light detected by the emission
light detector while changing a wavelength of the excitation light
irradiated onto the sample cell by the excitation light side
spectroscope, wherein the computer employs a plurality of
combinations of a range of wavelengths of the excitation light
dispersed by the excitation light side spectroscope and a range of
wavelengths of the emission light dispersed by the emission side
spectroscope.
8. The fluorescence detector for a liquid chromatograph according
to claim 7, wherein the computer calculates the three-dimensional
spectrum of the emission light intensity in the sample on the basis
of the set combinations of the range of wavelengths of the
excitation light dispersed by the excitation light side
spectroscope and the range of wavelengths of the emission light
dispersed by the emission side spectroscope.
Description
BACKGROUND
[0001] The present invention relates to a fluorescence
spectrophotometer, and a fluorescence detector for a liquid
chromatograph.
[0002] Fluorescence spectrophotometers are provided with a sample
cell that houses a sample, an excitation light side spectroscope
that disperses light generated from a light source, an emission
side spectroscope that disperses emission light generated when the
excitation light dispersed by the excitation light side
spectroscope is irradiated onto the sample, and a detector that
detects the emission light sent from the emission side
spectroscope.
[0003] As a method for determining the wavelength of emission light
so as to identify components of a sample, there has been proposed a
technique of respectively scanning the wavelength of excitation
light and the wavelength of emission light, obtaining a
three-dimensional spectrum on which the excitation light
wavelength, the emission light wavelength, and the emission light
intensity are plotted, comparing the three-dimensional spectrum
with a preliminarily-obtained three-dimensional spectrum in a
standard sample, and thereby determining the excitation light
wavelength and the emission light wavelength specific to the sample
(as is disclosed in Japanese Unexamined Patent Application
Publication No. JP-A-06-109542).
SUMMARY OF THE INVENTION
[0004] To obtain the three-dimensional spectrum in the
aforementioned conventional technique, for example, when a scanning
wavelength range is set to 300 nm to 800 nm, the emission light
intensity is detected by a detector while the emission light
wavelength is being scanned from 300 nm to 800 nm with the
excitation light wavelength being fixed to 300 nm. Subsequently,
the excitation light wavelength is increased by, for example, 10 nm
to 310 nm and fixed, and the emission light intensity is detected
by the detector while the emission light wavelength is being
scanned from 300 nm to 800 nm. The emission light intensity is
detected up to an excitation light wavelength of 800 nm in a
similar manner, to thereby obtain the three-dimensional spectrum of
the emission light intensity. The three-dimensional spectrum of the
emission light intensity in the sample can be obtained by measuring
the respective wavelengths in the excitation light wavelength range
while sequentially scanning the emission light wavelengths as
described above.
[0005] However, for example, when an operator intends to obtain
data in a given wavelength range instead of the above entire
wavelength range, it is necessary for the operator to input and set
a start wavelength and a stop wavelength for the wavelength
scanning with respect to each wavelength range since only the start
wavelength and the stop wavelength can be set in the conventional
apparatus, and it takes at least about one minute to obtain the
three-dimensional spectrum of the emission light intensity.
Accordingly, it takes a long time to perform all measurements
desired by the operator.
[0006] Since the sample is always irradiated with the excitation
light during the measurement of the three-dimensional spectrum,
there is also a risk that a sample with low chemical stability is
deteriorated or decomposed due to irradiation energy of the
excitation light when a measuring time is long.
[0007] Moreover, since the obtained data of the three-dimensional
spectrum includes the scanning wavelength of the excitation light,
the scanning wavelength of the emission light, and the emission
light intensity, the data has a large size, and a large load is
applied to an arithmetic unit that performs data processing, so
that a processing time is extended.
[0008] It is an object of the present invention to provide a
fluorescence spectrophotometer and a fluorescence detector for a
liquid chromatograph which shorten a time required for measuring a
three-dimensional spectrum in a sample.
[0009] To achieve the above object, an embodiment of the present
invention includes: a sample cell that houses a sample, the
components of which are analyzed; an excitation light side
spectroscope that irradiates excitation light with a predetermined
wavelength onto the sample cell; an emission side spectroscope that
disperses emission light from the sample cell by scanning a
predetermined range of wavelengths; an emission light detector that
detects the emission light from the emission side spectroscope; and
a computer that obtains a three-dimensional spectrum of an emission
light intensity in the sample on the basis of a wavelength and an
intensity of the emission light detected by the emission light
detector while changing a wavelength of the excitation light
irradiated onto the sample cell by the excitation light side
spectroscope, wherein the computer sets a plurality of combinations
of a range of wavelengths of the excitation light dispersed by the
excitation light side spectroscope and a range of wavelengths of
the emission light dispersed by the emission side spectroscope.
[0010] The present invention can provide a fluorescence
spectrophotometer and a fluorescence detector for a liquid
chromatograph which can shorten a time required for measuring a
three-dimensional spectrum in a sample.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 is a configuration diagram illustrating a main
configuration of a fluorescence spectrophotometer.
[0012] FIG. 2 is a screen view illustrating one example of a screen
for setting conditions for obtaining three-dimensional fluorescence
spectrum.
[0013] FIG. 3 is a graph showing a scanning region of the
excitation light wavelength and the emission light wavelength.
[0014] FIG. 4 is a graph showing a scanning region of the
excitation light wavelength and the emission light wavelength.
[0015] FIG. 5 is a configuration diagram illustrating a main
configuration of a liquid chromatograph.
DETAILED DESCRIPTION OF THE INVENTION
[0016] In the following, an embodiment of the present invention
will be described by reference to the drawings.
Embodiment
[0017] FIG. 1 is a configuration diagram illustrating a main
configuration of a fluorescence spectrophotometer. As shown in FIG.
1, a fluorescence spectrophotometer 100 is composed of a photometer
unit 110, a data processing unit 120, and an operation display unit
130. In the photometer unit 110, continuous light emitted from a
light source 111 is dispersed as monochromatic excitation light by
an excitation side spectroscope 112, and irradiated onto a
measurement sample installed on a sample installation section 115
through a beam splitter 113. The beam splitter 113 disperses a
portion of the excitation light, and a monitor detector 114
measures the intensity of the light, which is used for monitoring a
variation in the light intensity of the continuous light emitted
from the light source 111 and correcting the variation in the data
processing unit 120. Emission light is emitted from the measurement
sample due to the irradiation of the excitation light. The emitted
emission light is dispersed into monochromatic light by an emission
side spectroscope 116, detected by a detector 117, and transmitted
as an electric signal according to the light intensity of the
emission light. The intensity signal of the emission light
transmitted from the detector 117 is converted to a digital signal
via an A/D converter 121 of the data processing unit 120, and
introduced into a computer 122. The computer 122 stores the
obtained intensity signal of the emission light in a storage
section provided inside the computer 122 as data correlated with
the wavelength of the excitation light obtained by dispersing the
continuous light by the excitation side spectroscope 112 and the
wavelength of the emission light obtained by dispersing the
emission light by the emission side spectroscope 116. The data can
be also displayed on a display section 131 when an operator uses an
operation section 132 of the operation display unit 130.
[0018] The excitation side spectroscope 112 and the emission side
spectroscope 116 are respectively composed of a diffraction grating
that spectrally resolves incident light, and a slit that receives
the spectrally-resolved incident light and selectively extracts
light with a particular wavelength, i.e., monochromatic light from
the spectrally-resolved incident light. The wavelength of light to
be transmitted through the slit is determined by the position of
the slit for receiving the spectrally-resolved incident light. In a
normal spectroscope, the wavelength of light to be transmitted
through a slit is determined by fixing the position of the slit and
rotating a diffraction grating little by little. The diffraction
grating of the excitation side spectroscope 112 is rotated by an
excitation side pulse motor 118 via a gear or a cam. The
diffraction grating of the emission side spectroscope 116 is also
rotated by an emission side pulse motor 119 via a gear or a
cam.
[0019] When the operation section 132 of the operation display unit
130 instructs the photometer unit 110 to perform emission light
measurement of the measurement sample, the computer 122 of the data
processing unit 120 drives the excitation side pulse motor 118
according to a measuring program stored in the unillustrated
storage section. The diffraction grating of the excitation side
spectroscope 112 is thereby rotated, so that the wavelength of the
excitation light to be extracted by the excitation side
spectroscope 112, i.e., the wavelength of the dispersed
monochromatic light is set.
[0020] Similarly, the computer 122 of the data processing unit 120
drives the emission side pulse motor 119 according to the measuring
program stored in the unillustrated storage section. The
diffraction grating of the emission side spectroscope 116 is
thereby rotated, so that the wavelength of the emission light to be
extracted by the emission side spectroscope 116, i.e., the
wavelength of the dispersed monochromatic light is set. A
mechanical section that sets the wavelength of the monochromatic
light as described above is called wavelength drive system.
[0021] In the measurement of the fluorescence spectrophotometer, a
scanning range of the wavelength of the excitation light and a
scanning range of the wavelength of the emission light are set
first. FIG. 2 is a screen view illustrating one example of a screen
for setting three-dimensional spectrum obtaining conditions.
[0022] In the present embodiment, an instance in which an operator
predicts and sets a measuring range of the wavelength of the
excitation light and a measuring range of the wavelength of the
emission light on the basis of the type and the property of the
measurement sample is shown. Here, No. 1 shows an instance 1 in
which the wavelength of the excitation light ranges from 300 nm to
400 nm, and the wavelength of the emission light ranges from 300 nm
to 400 nm, and No. 2 shows an instance in which the wavelength of
the excitation light ranges from 500 nm to 700 nm, and the
wavelength of the emission light ranges from 700 nm to 800 nm. When
the measurement sample is a mixed sample containing a plurality of
components or when the measurement sample is predicted to have a
plurality of excitation light wavelengths or emission light
wavelengths according to the property of the measurement sample, a
plurality of combinations of scanning wavelength ranges can be set.
As described above, by employing a combination of a start
wavelength and a stop wavelength of each of the measuring range of
the wavelength of the excitation light and the measuring range of
the wavelength of the emission light as one set, a plurality of
sets of combinations can be set at the time of setting the
measuring conditions. The fluorescence spectrophotometer performs
automatic measurement according to the set contents, and stores the
obtained data of a three-dimensional spectrum in the unillustrated
storage section. In the embodiment of the present invention, the
wavelength range can be preliminarily-specified before measurement,
and the plurality of wavelength ranges can be automatically
measured, so that a measuring time can be significantly shortened
as compared to that in a conventional apparatus.
[0023] FIG. 3 is a graph showing a scanning region of the
excitation light wavelength and the emission light wavelength. A
horizontal axis represents an excitation light wavelength Ex, and a
vertical axis represents an emission light wavelength Em. When a
measurer intends to obtain only the emission light intensity in
wavelength scanning regions shown in FIG. 2, it is necessary for
the conventional apparatus to obtain the data of the emission light
intensity by scanning all of regions 301, 302, 303, and 304, that
is, scanning the wavelengths of the excitation light ranging from
300 nm to 700 nm, and scanning the wavelengths of the emission
light ranging from 300 nm to 800 nm.
[0024] Meanwhile, in the present embodiment, when the settings of
the wavelength scanning regions shown in FIG. 2 are input and
instructed, the computer 122 instructs the respective sections of
the photometer unit 110 to scan wavelengths only in the ranges of
the regions 303 and 304, and thereby obtain the data of the
emission light intensity therein.
[0025] Since the wavelength of the emission light is smaller than
the wavelength of the excitation light, only the region 303 in
which the excitation light wavelength is smaller than the emission
light wavelength out of a region enclosed by a wavelength range
having the wavelengths of the excitation light ranging from 300 nm
to 400 nm and a wavelength range having the wavelengths of the
emission light ranging from 300 nm to 400 nm in FIG. 3 is scanned
and the data thereof is obtained, so that the time is
shortened.
[0026] As described above, in the present embodiment, since any
wavelength region can be selectively specified as a wavelength
region whose data is desired to be obtained by an operator, such an
advantage that the time required for obtaining the data is
shortened, and the size of the obtained data is reduced as compared
to the conventional case can be obtained.
[0027] Also, the scanning of wavelengths is often performed per 10
nm, and the data of the emission light intensity in this case is
represented as a matrix per 10 nm on both the horizontal and
vertical axes and stored in the unillustrated storage section. At
this time, as for the data group stored in the storage section, a
value indicating that no data is provided since measurement has not
been performed, e.g., "zero" is written into the cells of the
matrices of the regions 301 and 302 whose wavelengths are not
scanned in FIG. 3. Accordingly, when the measurement data is
displayed on the display section 131, it is possible to display the
measurement data such that an unmeasured wavelength region is
apparent at first sight by using the fact that the cells into which
"zero" is written constitute the unmeasured wavelength region, and
the operator is thereby enabled to determine the necessity of
measurement in the unmeasured wavelength region.
[0028] FIG. 4 is a graph showing a scanning region of the
excitation light wavelength and the emission light wavelength in a
similar manner to FIG. 3. FIG. 4 differs from FIG. 3 in that
wavelengths in a region 405 out of the region 301 in which the
excitation light wavelength is larger than the emission light
wavelength are scanned. The case of the instance shown in FIG. 3
has the advantage that the time can be shortened since the
wavelengths in the region 405 are not scanned. However, in a case
of an apparatus in which such control as to operate the emission
side pulse motor 119 to change the wavelength for starting the
scanning of the emission light wavelength needs to be performed
with the excitation light wavelength and the emission light
wavelength being along the same line, a control program for the
emission side pulse motor 119 out of programs executed by the
computer 122 is rewritten.
[0029] Meanwhile, in the instance shown in FIG. 4, the scanning of
the emission light wavelength is started from 300 nm, and the data
is obtained only in a region 403. Therefore, only a timing of
obtaining the data executed by the computer 122 may be changed
without changing the control program for the emission side pulse
motor 119 out of the programs executed by the computer 122.
Accordingly, although the time required for the wavelength scanning
is slightly longer than that in the instance shown in FIG. 3, the
time can be significantly shortened as compared to the conventional
case in which the data is obtained by scanning a wavelength region
front surface.
[0030] As described above, with the embodiment of the present
invention, the fluorescence spectrophotometer which can shorten the
time required for measuring the three-dimensional spectrum in the
sample, prevent deterioration in the sample, and reduce the data
size can be obtained.
[0031] FIG. 5 is a configuration diagram illustrating a main
configuration of a liquid chromatograph. The liquid chromatograph
apparatus is an apparatus which separates components of a sample in
a separation column while transferring the sample by an eluent,
detects the sequentially transferred components by a detector, and
thereby analyzes the components of the sample. The configuration
shown in FIG. 5 will be described as one example thereof. The
eluent is transferred by a liquid transfer device 502 such as a
syringe pump from an eluent container 501 that stores the eluent
for carrying a sample. A given amount of sample is injected into
the eluent in a sample injection unit 503, and sent to a column
504. Since the flow speed differs depending on the type of the
component of the sample in the eluent due to the action of a
filling material filled inside a pipe of the column 504, the
separated components sequentially flow out of the column 504. The
sample components can be identified by detecting the emission light
wavelengths of the components by use of, for example, the
fluorescence spectrophotometer shown in FIG. 1 according to the
embodiment of the present invention as a detector 505 that detects
the components while causing the components to flow through a flow
cell as a sample cell, and thereby creating a three-dimensional
spectrum.
[0032] In a conventional detector, only start and end values can be
set for the scanning range of the excitation light wavelength and
the scanning range of the emission light wavelength. However, when
the fluorescence spectrophotometer according to the present
invention is used as the detector of the liquid chromatograph, the
time from the start to the end of the wavelength scanning is
shortened since it is possible to set and execute the scanning of
only wavelengths required by an operator and the obtaining of the
data. Accordingly, the number of wavelength scanning operations
performed while the components are passing through the flow cell is
increased even in an ultrafast liquid chromatograph in which the
components flow at high speed, and the component identification
accuracy is improved.
[0033] As described above, with the embodiment of the present
invention, the fluorescence detector for a liquid chromatograph
which can shorten the time required for measuring the
three-dimensional spectrum in the sample, and improve the component
identification accuracy can be obtained.
REFERENCE SIGNS LIST
[0034] 100 Fluorescence spectrophotometer, 110 Photometer unit, 111
Light source, 112 Excitation side spectroscope, 113 Beam splitter,
114 Monitor detector, 115 Sample installation section, 116 Emission
side spectroscope, 117 Detector, 118 Excitation side pulse motor,
119 Emission side pulse motor, 120 Data processing unit, 121 A/D
converter, 122 Computer, 130 Operation display unit, 131 Display
section, 132 Operation section, 501 Eluent container, 502 Liquid
transfer device, 503 Sample injection unit, 504 Column, 505
Detector
* * * * *